Picosecond Laser Processing of Photosensitive Glass for Generation of Biologically Relevant Microenvironments

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Article Picosecond Laser Processing of Photosensitive Glass for Generation of Biologically Relevant Microenvironments

Florin Jipa 1, Stefana Orobeti 1, Cristian Butnaru 1, Marian Zamﬁrescu 1, Emanuel Axente 1 , Felix Sima 1,2,* and Koji Sugioka 2,*

1 CETAL, National Institute for Laser, Plasma and Radiation Physics (INFLPR), 409 Atomistilor, RO-77125 Magurele, Romania; florin.jipa@inflpr.ro (F.J.); stefana.iosub@inflpr.ro (S.O.); cristian.butnaru@inflpr.ro (C.B.); marian.zamfirescu@inflpr.ro (M.Z.); emanuel.axente@inflpr.ro (E.A.) 2 RIKEN Center for Advanced Photonics, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan * Correspondence: felix.sima@inflpr.ro (F.S.); [email protected] (K.S.); Tel.: +40-21-457-4550 (F.S.)

Received: 28 October 2020; Accepted: 12 December 2020; Published: 15 December 2020

Abstract: Various material processing techniques have been proposed for fabrication of smart surfaces that can modulate cellular behavior and address specific clinical issues. Among them, laser-based technologies have attracted growing interest due to processing versatility. Latest development of ultrashort pulse lasers with pulse widths from several tens of femtoseconds (fs) to several picoseconds (ps) allows clean microfabrication of a variety of materials at micro- and nanoscale both at surface and in volume. In this study, we addressed the possibility of 3D microfabrication of photosensitive glass (PG) by high repetition rate ps laser-assisted etching (PLAE) to improve the fabrication efficiency for the development of useful tools to be used for specific biological applications. Microfluidic structures fabricated by PLAE should provide the flow aspects, 3D characteristics, and possibility of producing functional structures to achieve the biologically relevant microenvironments. Specifically, the microfluidic structures could induce cellular chemotaxis over extended periods in diffusion-based gradient media. More importantly, the 3D characteristics could reproduce capillaries for in vitro testing of relevant organ models. Single cell trapping and analysis by using the fabricated microfluidic structures are also essential for understanding individual cell behavior within the same population. To this end, this paper demonstrates: (1) generation of 3D structures in glass volume or on surface for fabrication of microfluidic channels, (2) subtractive 3D surface patterning to create patterned molds in a controlled manor for casting polydimethylsiloxane (PDMS) structures and developing single cell microchambers, and (3) designing glass photo-masks to be used for sequel additive patterning of biocompatible nanomaterials with controlled shapes, sizes, and periodicity. Mesenchymal stem cells grown on laser-processed glass surfaces revealed no sign of cytotoxicity, while a collagen thin coating improved cellular adhesion.

Keywords: picosecond laser-assisted etching; photosensitive glass; 3D microfluidics; PDMS; microchambers; photo-masks; biomimetics

1. Introduction Biochips, such as microﬂuidic, lab-on-a-chip, and organ-on-a-chip devices, and micro total analysis systems have opened a new door for medical and biological investigations due to capability of generating biologically relevant microenvironments and potential of single cell analysis [1,2]. Generally, shape-controlled micropatterns grown on solid substrates by additive manufacturing are adequate for the generation of biomimetic cell-instructive environments, able to modulate speciﬁc cell responses for applications in tissue engineering, regenerative medicine, or cancer therapy. These surfaces and cell interfaces are needed for the next generation of biomaterials [3]. For example, in the cancer therapy,

Appl. Sci. 2020, 10, 8947; doi:10.3390/app10248947 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 8947 2 of 17 studies have shown that tumor cells may exhibit ‘plasticity’ in response to microenvironmental cues [4]. Interfacial geometry that dictates cancer cell tumorigenicity has revealed how the characteristics of micropatterns (e.g., size, shape, and perimeter characteristics) influence a population of tumor cells that can further guide cancer cells toward a stem cell-like state [5]. A clinical biochip consisting of more than 1000-nanoliter-sized microchambers was developed for single immune cell investigation [6]. The proteins secreted by individual cells were then captured by antibody stripes followed by multiplexed fluorescent detection. Such bioplatform proved successful ex vivo quantification of the polyfunctional diversity of tumor antigen-specific T lymphocytes. New biochip platforms consisting of microwell arrays were recently proposed for cell-based immunological studies with various applications related to immune cell secretion, cytotoxicity, migration, phenotyping, and immune cell engineering [7]. In addition, high-throughput cell trapping assays with single cell accuracy were efficiently developed into microfluidic lab-on-chip systems [8]. Meanwhile, lasers are increasingly getting important as tools for biomaterial modification or fabrication of biochips. The main advantages of laser techniques in comparison to other approaches are extensively addressed in the literature [9,10]. One can mention the high processing versatility of a variety of materials together with fast, clean, flexible, and controllable microfabrication capability. During the last decade, laser technologies were explored in the field of biomimetics with new cutting-edge applications [11]. The state of the art of “Laser Technology in Biomimetics” covering a broad spectrum of materials, methodologies, and applications was comprehensively reported in [12]. This book covers aspects of laser–matter interaction of nanosecond pulsed lasers for 2D patterning using well-known deposition techniques, such as pulsed laser deposition (PLD) and matrix-assisted pulsed laser evaporation (MAPLE) for material coating and ultrashort laser pulses for 3D modification of materials. PLD and MAPLE employ pulsed laser beams, typically nanosecond laser pulses, to ablate solid targets in a vacuum. Then, the ablated material is deposited onto substrates opposed to the targets to form thin coatings. Different from PLD, which is used for inorganic coatings only, the MAPLE process employs milder conditions (laser energies one order of magnitude lower) adequate for coating any inorganic, organic, and hybrid materials with high versatility. Thus, the safe transfer and deposition of delicate compounds such as proteins and biopolymers are possible without affecting stability and deteriorating functionalities [11]. In addition, graphene oxide (GO) and GO nanocolloids (GON) can be deposited by MAPLE, which are very challenging matrices for drug release to target cancer cells. It has been very recently found that the MAPLE process provides safe concentration windows of GON for five different human melanoma cell lines as well as for nontransformed melanocytes and human dermal fibroblasts [13]. A specific feature of laser–matter interaction, in particular when using ultrashort pulse lasers with pulse widths of several tens of femtosecond (fs) to several picoseconds (ps), offers the possibility to precisely control three-dimensional (3D) micro-/nanofabrication in transparent materials [14–20]. Indeed, tightly focused ultrashort pulse lasers can achieve very high peak intensities ( 10 TW cm 2), ≈ − resulting in strong nonlinear absorption phenomena in transparent materials to allow localized modification in volume with high precision [21]. The fundamental mechanisms of fs laser pulse interaction with the transparent materials were extensively explored in the literature [15,17,19,21]. Some of the involved processes are competitive at different time scales and are strongly dependent not only on the laser irradiation parameters (pulse energy, pulse duration, laser wavelength, repetition rate, and focusing optics) but also on the physical-chemical properties of materials processed. The extensive studies of the laser–matter interaction together with new development of high-precision 3D CAD-CAM (computer-aided design/computer-aided manufacturing) stages potentiate the unique characteristics of ultrashort pulse lasers for 3D micro- and nanofabrication of the diverse materials. Advanced setups consisting of ultrashort pulse lasers and high-precision 3D translation stages have been developed for selective and precise generation of complex 3D microstructures embedded inside photosensitive glasses (PGs). The process is known as fs laser-assisted etching (FLAE) and Appl. Sci. 2020, 10, 8947 3 of 17 was continuously optimized for concrete applications [22–27]. It begins with laser direct writing (LDW) of PG in which the stage scanning speed, focusing position, and laser power are adjusted to achieve the desired 3D shape structure. An initial annealing treatment follows the laser irradiation, by which Ag atoms generated by the laser irradiation are clustering to form nuclei for the growth of a crystalline phase of lithium metasilicate at the laser-exposed regions. The wet chemical etching using hydrofluoric (HF) acid solution is then applied for selective removal of the crystalline phase to create 3D hollow structures. Consequently, microfluidic platforms with scale-down (µm) and scale-up (mm) configurations can be achieved. A typical PG used with FLAE is commercially available Foturan glass. It exhibits high chemical stability, high transparency for optical interrogation, and biocompatibility. Advanced laser processing protocols of Foturan glass were described in details in [22,28–30]. Pioneering FLAE of Foturan glass and the fabrication of complex 3D microfluidic structures with a high spatial resolution were reported by Masuda et al. [22]. Microstructures fabricated in Foturan glass proved to be useful for many applications due to good properties in terms of surface smoothness of created 3D structures [25,31], capability of selective metallization [32–34], and good process efficiency [35]. Specifically, 3D microfluidic structures fabricated in Foturan glass by FLAE were successfully applied to fabricate nanoaquariums for microorganisms’ studies (e.g., 3D monitoring of flagellum motion of Euglena gracilis [30] or gliding mechanism of Phormidium cyanobacteria [36]). A very recent study evidenced the possibility to generate submicrometer 3D channels inside Foturan by FLAE, for mimicking the intravasation–extravasation processes during chemotaxis-free cancer cells’ migration inside confined nanospaces [37]. An alternative material used for microfluidic assays for multiple biological applications is a soft polymer polydimethylsiloxane (PDMS) due to its biocompatibility, transparency, high molding properties, tunable mechanical features, and low cost [38,39]. However, it has been shown that biomolecule adsorption by PDMS is a current limitation, especially in case of biological testing such as particular drug response assays [40]. Additionally, fabrication of complex 3D structures is still challenging with this material. On the other hand, glass seems to be a more appropriate material for some applications, in particular when long-time cellular observation or high-resolution cell imaging is required. Thus, glass biochips fabricated by FLAE represent innovative platforms that are extensively explored to advance not only fundamental studies, but also clinical applications in nanomedicine for overcoming the limitations of traditional biomedical approaches [7,41,42]. They can further represent miniaturized micropatterned devices, including but not limited to microfluidics, microwell chips, and microarrays proposed for specific bioassays. Their main advantages as compared with conventional bioassays are related to the use of low sample volumes; accurate control over sample manipulation and analyses; high spatial resolution down to a single cell, high-throughput screening; and broad spectrum of in situ measurements [7]. In the field of ultrafast laser processing for the development of biochips, fs lasers are mostly used as described above, and few works have been done with ps lasers in spite of the fact that the ps lasers offer some advantages such as higher power, higher reliability, and higher stability, which are essential for practical use. We recently introduced the possibility of precise microfabrication of PG volume by high repetition rate ps laser-assisted etching (PLAE) [28]. Along with the determination of critical irradiation doses and etching rates for glass processing with either ultraviolet (355 nm) or visible (532 nm) laser wavelengths, the study revealed the very fast and controlled synthesis of embedded 3D microfluidic channels with high aspect ratio, reduced processing time, and lower fabrication cost. The main advantage of PLAE over FLAE was found to be the short laser processing interval, up to 10 times faster, which is beneficial for fabrication of large-area (cm sizes) biochips, while keeping a microscale processing resolution. In the present study, we evaluated three distinct approaches of applying PLAE to large-area PG processing: (1) microfabrication of complex 3D interconnected glass channels, (2) laser surface patterning to fabricate PG molds for PDMS replication of numerous microchambers, and (3) PG masks consisting of microholes with different sizes to be used for the synthesis of nanomaterial patterns by Appl. Sci. 2020, 10, x FOR PEER REVIEW 4 of 18 Appl. Sci. 2020, 10, 8947 4 of 17 masks consisting of microholes with different sizes to be used for the synthesis of nanomaterial patternssubsequent by subsequent additive processes. additive processes. The customized The cu micropatternsstomized micropatterns were evaluated, were evaluated, and the fabricated and the fabricatedmicrofluidic microfluidic devices were devices applied were for applied cell cultures for cell and cultures single and cell single immobilization. cell immobilization.

2.2. Materials Materials and and Methods Methods LaserLaser Processing. Processing. All All experiments experiments were were performed performed using using Foturan Foturan PG PG (commercially (commercially available available fromfrom Schott). Schott). Foturan Foturan is isa aphoto-structurable photo-structurable lithi lithiumum alumino-silicate alumino-silicate glass glass that that accommodates accommodates small small amountsamounts of of Ag Ag2O2O (0 (0.05–0.15%).05–0.15%) and and Ce Ce2O32O3 (0. (0.01–0.04%),01–0.04%), primarily primarily designed designed for for the the UV UV light-induced light-induced selectiveselective microstructuring. microstructuring. TheThe protocol protocol for for obtaining obtaining free-form free-form shaped shaped micr microstructuresostructures in Foturan in Foturan glass glass using using PLAE PLAE is schematicallyis schematically depicted depicted in Figure in Figure 1. LDW1. LDWwas carrie wasd carried out using out a using ps laser a ps source laser (Coherent, source (Coherent, model Lumeramodel LumeraHyperRapid HyperRapid 50), delivering 50), delivering pulses of pulses about of 10-ps about duration 10-ps duration at 500-kHz at 500-kHz repetition repetition rate. The rate. large-scaleThe large-scale area microfluidic area microﬂuidic structures structures and andPG PGmolds molds were were fabricated fabricated using using visible visible (VIS—532 (VIS—532 nm) nm) psps laser, laser, which which proved proved to to be more appropriate forfor aa precise precise and and controlled controlled volume volume processing, processing, while while UV UV(355 (355 nm) nm) laser laser pulses pulses were were employed employed for for fabricationfabrication ofof photo-masks, sinc sincee it it was was experimentally experimentally foundfound that that the the ps ps laser laser was was more more adequate adequate for for efficient eﬃcient and and rapid rapid formation formation of of through-holes through-holes in in bulk bulk glass.glass. Laser Laser power power was was adjusted adjusted between between 30–50 30–50 mW mW in in case case of of UV UV and and 500–600 500–600 mW mW for for VIS VIS laser laser pulses.pulses. The The laser laser beam beam was was focused focused onto onto the the sample sample surface surface using using an an aspheric aspheric lens lens of of 15-mm 15-mm focal focal lengthlength and and 0.5 0.5 numerical numerical aperture aperture to to generate generate a circularcircular spotspot ofof aboutabout 4 4µ µmm in in diameter diameter (calculated (calculated by byw w= =2f 2fλMλM2/π2/D,πD, where where w w is theis the minimum minimum beam beam waist waist radius, radius, f is f ais focal a focal distance distance of theof the focusing focusing lens, lens,M is M a is quality a quality beam beam factor, factor, D is D a is laser a laser beam beam diameter, diameter, and andλ is λ the is wavelengththe wavelength of the of laserthe laser used used [43]). [43]).The writingThe writing speed speed was varied was varied from 0.1 from to 1 0.1 mm to/s depending1 mm/s depending on the pattern on the dimensions pattern dimensions and geometrical and geometricalfeatures. The features. samples The were samples placed were on a placed customized on a customized LDW processing LDW platformprocessing consisting platform of consisting motorized oftranslation motorized stagestranslation (PlanarDL stages Aerotech),(PlanarDL providingAerotech), a providing spatial resolution a spatial ofresolution a few tens of ofa few nanometers tens of nanometers(Figure1a). The(Figure experiments 1a). The wereexperiments carried outwere in acarri cleaned room, out in in a air, clean at atmospheric room, in air, pressure, at atmospheric and were pressure,in-situ monitored and were within-situ a charge-coupledmonitored with devicea charge-coupled (CCD) camera device (Thorlabs). (CCD) camera (Thorlabs).

FigureFigure 1. 1. SchematicSchematic of of the the PLAE PLAE (picosecond (picosecond laser-assisted laser-assisted etching) etching) process: process: (a ()a laser) laser direct direct writing writing followedfollowed by by (b ()b thermal) thermal treatment treatment and and (c ()c )chemical chemical etching etching in in HF HF (hydrofluoric (hydroﬂuoric acid). acid).

InIn a a subsequentsubsequent step step (Figure (Figur1b),e the1b), laser-exposed the laser-exposed glass samples glass weresamples annealed were in aannealed programmable in a programmablefurnace (Carbolite, furnace model (Carbolite, MTF M1238-250). model MTF First,M1238-250 controlled). First, heating controlled with heating a slope with of 5 ◦ aC slope/min upof 5 to °C/min500 ◦C up was to performed.500 °C was performed. Then the temperature Then the temper was keptature constant was kept for constant 1 h in order for 1 toh in cause order di fftousion cause of diffusionprecipitated of precipitated Ag atoms toAg grow atoms Ag to nanoclusters. grow Ag nanoclusters. Next, the temperature Next, the temperature was increased was again increased with a againslope with of 3 a◦C slope/min of up 3 to°C/min 605 ◦C up and to kept605 °C constant and kept for constant another for hour another to induce hour the to formation induce the of formation crystalline oflithium crystalline metasilicate lithium phasemetasilicate around phase the grown around Ag the nanoclusters, grown Ag which nanoclusters acted as, which nuclei. acted The latent as nuclei. image Theformed latent during image theformed laser during direct writingthe laser process direct writing was then process developed was then by thedeveloped thermal by treatment the thermal to be treatment to be visible. By changing the laser fluence and scanning speed one may thus modify the Appl. Sci. 2020, 10, 8947 5 of 17 Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 18 visible.glass Byand changing evaluate the tracing laser line fluence thicknesses and scanning (Figure speed 2). The one pulse may overlap thus modify for all the cases glass can and be evaluate estimated tracingto more line than thicknesses 99.9%. This (Figure evaluation2). The pulseallows overlap choosin forg further all cases laser can beparameters estimated for to the more glass than processing 99.9%. Thisfor evaluation specific needs, allows e.g., choosing thinner further lines for laser higher-resolution parameters for processing the glass processing or thicker forlines specific for larger-area needs, e.g.,modification. thinner lines In for our higher-resolution case we found processingthreshold line or thickerwidths lines of about for larger-area 4 μm in case modification. of UV laser In pulses our caseand we of found 5.5 μm threshold in case of line VIS, widths which ofcan about increase 4 µm with in case increasing of UVlaser laser pulsesfluence and and of decreasing 5.5 µm in casescanning of VIS,speed which (Figure can increase 2b,d). with increasing laser fluence and decreasing scanning speed (Figure2b,d).

FigureFigure 2. Evaluation2. Evaluation of lineof line widths’ widths’ function function of of laser laser ﬂuence fluence and and scanning scanning speed speed in in case case of of UV UV (a, b(a),b) andand VIS VIS laser laser irradiation, irradiation, respectively respectively (c, d(c).,d Recorded). Recorded optical optical images images (a, c(a), andc) and corresponding corresponding plotted plotted graphsgraphs (b, d(b), ared) are depicted. depicted.

In the last step, the glass samples were submitted to chemical etching in an 8% HF solution under In the last step, the glass samples were submitted to chemical etching in an 8% HF solution under the ultrasonic condition (Figure1c). During the wet etching process, the grown crystalline phase was the ultrasonic condition (Figure 1c). During the wet etching process, the grown crystalline phase was selectively removed, since it had higher etching rate as compared with laser-unexposed regions [18] selectively removed, since it had higher etching rate as compared with laser-unexposed regions [18] Microstructures corresponding to the laser written trajectory were then produced in Foturan glass. Microstructures corresponding to the laser written trajectory were then produced in Foturan glass. The second annealing treatment at 635 C was further employed for surface smoothening. The second annealing treatment at 635◦ °C was further employed for surface smoothening. MAPLE processing: MAPLE experiments were performed inside a stainless-steel reaction chamber. MAPLE processing: MAPLE experiments were performed inside a stainless-steel reaction An UV KrF* excimer laser source (Lambda Physics Coherent, COMPexPro 205) delivering pulses at a chamber. An UV KrF* excimer laser source (Lambda Physics Coherent, COMPexPro 205) delivering wavelength of λ = 248 nm, with a duration of τFWHM = 25 ns (FWHM-Full Width at Half Maximum), pulses at a wavelength of λ = 248 nm, with a duration of τFWHM = 25 ns (FWHM-Full Width at Half and operated at a repetition rate of 5 Hz was used for thin film deposition through PG masks. In one Maximum), and operated at a repetition rate of 5 Hz was used for thin film deposition through PG experiment, a cryogenic target consisting of organic carbon precursors (phloroglucinol/glyoxylic acid masks. In one experiment, a cryogenic target consisting of organic carbon precursors and the template Pluronic F127 dissolved in a chloroform/ethanol mixture) was frozen in liquid (phloroglucinol/glyoxylic acid and the template Pluronic F127 dissolved in a chloroform/ethanol nitrogen. Three thousand pulses of 4 J cm 2 incident laser fluence were subsequently applied in a mixture) was frozen in liquid nitrogen. Three− thousand pulses of 4 J cm−2 incident laser fluence were dynamic pressure of ~5 10 2 mbar for deposition. In another experiment we used a target prepared subsequently applied× in a− dynamic pressure of ~5 × 10−2 mbar for deposition. In another experiment by immersing an aqueous solution containing graphene oxide nanocolloids (GON, 2 mg/mL Sigma we used a target prepared by immersing an aqueous solution containing graphene oxide Aldrich) in liquid nitrogen. We applied 25,000 pulses of <1 J cm 2 incident laser fluence, in a dynamic nanocolloids (GON, 2 mg/mL Sigma Aldrich) in liquid nitrogen.− We applied 25,000 pulses of <1 J cm−2 pressure of 2 10 2 mbar for the synthesis of the micropatterns. The structures were grown on silicon incident laser∼ × fluence,− in a dynamic pressure of ∼2 × 10−2 mbar for the synthesis of the micropatterns. substrates placed parallel to the target surface at separation distances of 4–5 cm. The structures were grown on silicon substrates placed parallel to the target surface at separation distances of 4–5 cm. Appl. Sci. 2020, 10, 8947 6 of 17

The characterization of the samples was performed by optical microscopy with a transmission mode (Leica, model DM4000 B Led). Scanning electron microscopy (SEM) investigation was carried out with an FEI Co. microscope (Inspect S model). In vitro tests, reagents, and antibodies: Mouse monoclonal anti-vinculin antibody was purchased from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany (#V9131), and goat anti-mouse IgG-coupled Alexa Flour 594 was purchased from Invitrogen (Thermo Fisher Scientific) (#A11005). Most cell culture reagents used in this study were purchased from Gibco (Fisher Scientific UK Ltd, Loughborough, UK): Penicillin-Streptomycin (#15140-122), Dulbecco’s modified Eagle’s medium (DMEM, #21885-025), Fetal Bovine Serum (#10270-106), and Glutamax (#35050-061). Triton X-100 (#T8787) and phosphate buffered saline tablets (#P4417-100TAB) were purchased from Sigma. Bovine Serum Albumin (BSA) was from VWRTM company (#422361V) (VWR International GmbH, Wien, Austria). ProLong Diamond Antifade Mountant was purchased from Invitrogen (#P36961). Cell culture: Primary human bone marrow-derived mesenchymal stem cells (MSC), a kind gift from the Institute of Biochemistry of Romanian Academy (Dr. Livia Sima), were used for experimental procedures in this study. They were grown in DMEM with 10% FBS (Fetal Bovine Serum), 1% Penicillin/Streptomycin (Pen-Strep) and 0.5 mM Glutamax. All cells were maintained in a humidified atmosphere at 37 ◦C and in the presence of 5% CO2. Sterilization: The Foturan glass samples and coverslips were autoclaved for 30 min at 121 ◦C, followed by exposure to UV for 30 min prior to use for any experimental procedures. Immunofluorescence microscopy: The surfaces of some coverslips and Foturan glass were coated with collagen (rat type I) for 30 min, followed by two times washing with PBS (Phosphate-Buffered Saline). The cells were then trypsinized and counted, and 3000 cells were seeded per sample. One hour or 24 h after seeding, cells were fixed using 1% PFA for 15 min at room temperature (RT), followed by permeabilization with 0.1% Triton X-100 for 5 min at RT. After permeabilization, the samples were blocked with 5% BSA for 1 h at RT and then incubated with primary antibody against vinculin (1:1000 dilution) in 5% BSA overnight at 4 ◦C. The next day, the Alexa Flour 594 conjugated secondary antibody and Alexa Fluor 488 conjugated Phalloidin were added in 5% BSA for 2 h at RT. The nuclei were stained with Hoechst in 5% BSA, 1:20,000 for 3 min at RT. After each step, PBS washes were performed three times for 5 min each. The samples were mounted using Prolong Diamond Antifade. Alternatively, for nuclei staining, we also mounted the samples by Vectashield with 40,6-diamidino-2-phenylindole DAPI (Vector Laboratories, Burlingame, CA, USA). Images were acquired using a Leica DM4000 B LED with the 20 magnification objective. × Acquired images were processed using ImageJ software. Colocalization analysis was performed using the ImageJ JACoP plugin. The images were split into separate channels and used for threshold processing. The analysis was performed in one experiment and the total number of fields analyzed is indicated in the figure legends. The obtained values were imported into GraphPad Prism 6 software for graphical representation. Statistical analysis: Data sets were analyzed using unpaired two-tailed Student’s t-test (with Welch correction). Mean values, SEM, and statistics were calculated with Prism6 (GraphPad software). Results with a p value less than 0.05 were considered to be significant as indicated in the figure legends. No criteria of inclusion or exclusion of data were used in this study.

3. Results and Discussion Ultrashort pulse laser processing of transparent Foturan glass with ps pulses was rarely addressed in the literatures. Veiko et al. [44] reported the possibility to modify Foturan glass by a ps Nd:YAG (neodymium-doped yttrium aluminum garnet (Nd:Y3Al5O12)) laser source delivering pulses of 30-ps duration at 532-nm wavelength and a repetition rate of 10 Hz. The same group showed the fabrication of 3D channels in Foturan bulk by HF etching of crystallized regions developed by annealing using a CO2 laser treatment [45]. Appl. Sci. 2020, 10, 8947 7 of 17

We recently proposed the PLAE technique as an alternative to FLAE [28] by demonstrating the successful fabrication of basic 3D embedded microchannels. Ps lasers could then provide advantage over fs lasers for large-area processing of some transparent materials both on surface and in volume. Higher average power and longer pulse width can modify more volume per pulse by depositing larger energy in the material. Specifically, lower peak intensity associated with the longer pulse width allowed us to irradiate the glass with higher energy pulse without generation of damage, which increases electron density and, thus, the precipitation of more Ag atoms. The larger amount of Ag atoms was then generated, which allowed us to more efficiently develop a larger glass crystalline area by the annealing treatment. Thus, the processing times can be shortened for large-area modification as compared with fs lasers. The developed area can be finally removed by HF chemical etching. In a comparison of fabricating a comparable structure, irradiation time was approximately 1.5 h for laser pulses of 360 fs at 522 nm (2 mm/s scanning speed and 250 KHz laser repetition rate), while approximately 10 min only were necessary for 10-ps laser pulses at 532 nm (a 0.9-mm/s scanning speed and 500-KHz laser repetition rate). The shortened irradiation time for the ps laser is mostly due to the reduced number of planes scanned using line-by-line laser irradiation required to cover the desired modified volume. In the ps case, the number of planes was significantly reduced as compared with fs processing due to the larger modified volume per pulse. A more efficient process was experimentally achieved with UV pulses, since the critical dose was one order of magnitude smaller than that of VIS pulses (laser power conversion efficiencies from the fundamental beam were 1:2 for VIS and 1:3 for UV) [28].

3.1. Fabrication of Large-Scale Microfluidic Channels in PG In this section, we report on the possibility to generate complex structures on PG surfaces for fabricating open, large-scale area microfluidic devices with central cell culture chambers (Figure3a,b). Final microfluidic chip was composed of two parts: (1) an open structure fabricated on PG surface by VIS-PLAE and (2) a PDMS sheet that sealed the fabricated structures on PG using the bounding process by activating their surfaces in oxygen plasma. The fabricated structure on PG consisted of cross-shaped microfluidic channels with microreservoirs at each end and a microchamber at the crossed region. In our design, the widths of all microfluidic channels were set to 0.3 mm while the central microchamber was 2 2 mm2 (Figure3a, corresponding to a laser-irradiated sample followed by × annealing treatment). It is worth to mention that the entire laser scanning process took 25 min only, at 1-mm/s scanning speed and 10-µm pitch between each scanning line. The open microstructures resulted from the selective glass removal induced by chemical etching are shown in Figure3b. Magnified images of the microchannel parts from Figure3a,b are presented in Figure3d,e, respectively. Due to the limited etching selectivity in HF between the laser-irradiated and nonirradiated regions, the actual lateral dimensions of obtained structures were larger than the initially set ones, i.e., the laser-irradiated regions. The widening depended on the etching time due to the etching rate of 1-µm/minute for the nonirradiated regions. Similarly, the depth of the channel can be controlled by the etching time. For example, one can get a 10-µm-deep channel in only 1 min or a 300-µm-deep channel in half an hour by taking account of the depth of modified region and the etching selectivity between nonirradiated and irradiated regions. Even though the roughness of the channel surface was significant after the etching, the second annealing treatment can well work to reduce the roughness to nanometer scale [25]. In our design, three of the microreservoirs acted as lateral inlets and the other one, as lateral outlet, while the microchamber was used for cell culture (Figure3c,f). Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 18

culture media or even drugs by a computer-controlled valve with sequential injection (Figure 3c,f). In a preliminary experiment we combined complete cell media (with serum), incomplete cell media Appl. Sci.(without2020, 10 serum),, 8947 and cell media supplemented with growth factors by serial perfusing through the 8 of 17 three inlets intermittently and monitored the response of mesenchymal stem cells (MSCs) over time.

FigureFigure 3. Microfluidic 3. Microfluidic chip chip fabricated fabricated byby PLAEPLAE in in Fotu Foturanran glass. glass. Photographic Photographic images images of the ofentire the entire structurestructure (a) after (a) after laser laser scanning scanning and and annealing,annealing, (b (b) )after after chemical chemical etching etching in HF, in and HF, ( andc) developed (c) developed chip (1.5chip (1.51.5 × cm 1.52 )cm connected2) connected to perfusionto perfusion system system that that cancan switchswitch between three three different different reagents reagents and × providesand provides a continuous a continuous flow; ( dflow;–f) detailed(d,e,f) detailed magnified magnified images images from from the the regions regions highlighted highlighted inin dotted squaresdotted in ( asquares–c), respectively. in (a–c), respectively.

A3.2. perfusion Fabrication system of Myriad (Elveflow, of Microc France)hambers in was PDMS coupled by Using to thePG fabricatedMolds chip using dedicated metallic connectorsStudies glued showed with the that lateral when inletscompared and with outlets conventi by SU-8onal photoresist.methods, the Theuse of setup microwell consisted biochips of several elements:in bio-nanotechnologies a pulsation-free could pressure result controller, in 100- to 1000-fold flow sensors, reduction tubing, in the andnumber reservoirs of cells required dedicated for to cell cultures.analyses A detailed due to great image reduction of the of microfluidic microchamber chip volume, during down flow to monitoring<1 nL [7]. is presented in Figure3f. Thus, a 3DWe biomimetic advance herein microenvironment the possibility to couldfabricate be up created to 10,000 in themicrochambers central chamber in PDMS for in cell a single growth and step based on a PG mold processed by PLAE. The protocol is schematically depicted in Figure 4. First, visualization, which can be associated with controlled flow aspects allowing a scale up-scale down we applied PLAE in order to create 3D pillars in PG after the chemical etching of laser-exposed approachregions. of theIn particular, device. Indeed, by scanning the inletstransversal were line easilys onto connected PG surface, to one the can perfusing design a systemchess-like while board channel dimensionswith square could dimensions be downsized tailored to by hundreds the CAD of (c micronsomputer-aided (Figure design)3d,e) orprogram smaller, by for a maskless specific cases. In addition,process independent(Figure 5a,b). controlThe laser-irradiated can be achieved regions for were inlet designed channels so from as to 1 todevelop 3 to mix desired any combinationdepth of fluidsdimensions. that also Specifically, allows perfusion one can irradiate switches either between with a multilayer different configuration culture media if deep or channels even drugs are by a computer-controlledrequired or with a valvesingle-layer with configuration sequential injection for superficial (Figure structuring.3c,f). In Similarly a preliminary to Section experiment 3.1., the we combinedlaser irradiation complete process cell media is followed (with serum), by annealing incomplete treatment cell atmedia 605 °C (withoutand chemical serum), etching and incell HF media solution. Figure 5c,d shows the exposed PG after annealing treatment where the brown color supplemented with growth factors by serial perfusing through the three inlets intermittently and represents modified PG and the white areas are the unmodified square structures. During the monitoredchemical the etching response the modified of mesenchymal PG was selectively stem cells re (MSCs)moved so over that time.pillars could be created from the unmodified square regions whose heights can be controlled with the etching time. The etching rate 3.2. Fabrication of Myriad of Microchambers in PDMS by Using PG Molds was constant regardless of times and depths since the sufficiently deep structures were modified and Studiesthe applied showed laser dose that was when kept compared constant within with th conventionale modified structures. methods, We, the thus, use obtained of microwell PG molds biochips 3 3 in bio-nanotechnologieswith 2500 pillars of 150 could × 150 result× 60 μm in 100-or even to 1000-foldwith 10,000 reduction pillars of 50 in × the50 × number 30 μm (length of cells × width required for × height). We used the fabricated PG molds to obtain replicas in PDMS, by which the pillar-prints analyses due to great reduction of microchamber volume, down to <1 nL [7]. were transcribed, i.e., microchambers with the same X-Y dimensions of glass pillars and depth Wecorresponding advance herein to the glass the possibility pillar height. to All fabricate processes up are to schematically 10,000 microchambers shown in Figure in PDMS 4. in a single step based on a PG mold processed by PLAE. The protocol is schematically depicted in Figure4. First, we applied PLAE in order to create 3D pillars in PG after the chemical etching of laser-exposed regions. In particular, by scanning transversal lines onto PG surface, one can design a chess-like board with square dimensions tailored by the CAD (computer-aided design) program by a maskless process (Figure5a,b). The laser-irradiated regions were designed so as to develop desired depth dimensions. Specifically, one can irradiate either with a multilayer configuration if deep channels are required or with a single-layer configuration for superficial structuring. Similarly to Section 3.1., the laser irradiation process is followed by annealing treatment at 605 ◦C and chemical etching in HF solution. Figure5c,d shows the exposed PG after annealing treatment where the brown color represents Appl. Sci. 2020, 10, 8947 9 of 17 modified PG and the white areas are the unmodified square structures. During the chemical etching the modified PG was selectively removed so that pillars could be created from the unmodified square regions whose heights can be controlled with the etching time. The etching rate was constant regardless of times and depths since the sufficiently deep structures were modified and the applied laser dose was kept constant within the modified structures. We, thus, obtained PG molds with 2500 pillars of 150 150 60 µm3 or even with 10,000 pillars of 50 50 30 µm3 (length width height). We used × × × × × × the fabricated PG molds to obtain replicas in PDMS, by which the pillar-prints were transcribed, i.e., microchambers with the same X-Y dimensions of glass pillars and depth corresponding to the glass pillarAppl. Sci. height. 2020, 10, All x FOR processes PEER REVIEW are schematically shown in Figure4. 9 of 18

Figure 4. Schematic of the fabrication procedure of the PG (photosensitive glass) mold and of 3D microchambersFigure 4. inSchematic PDMS. Theof the mold fabrication was obtained procedure after ofthe the PG PG processing (photosensitive protocol glass) by mold PLAE, and described of 3D in microchambers in PDMS. The mold was obtained after the PG processing protocol by PLAE, Section2. The PDMS replica was obtained after 24 h drying at room temperature upon separating the Appl. Sci.described 2020, 10, inx FOR Section PEER 2. REVIEW The PDMS replica was obtained after 24 h drying at room temperature upon10 of 18 PDMSseparating (Polydimethylsiloxane) the PDMS (Polydim fromethylsiloxane) the mold. from the mold.

Sketches and photographs of PG after the laser irradiation and the annealing treatment are shown in Figure 5a,b, while optical images of the exposed patterns (single-layer laser scanning) are visualized in Figure 5c,d. Similarly, Figure 5e,f depicts the mold sketch and photograph after chemical etching and Figure 5g,h presents magnified images of Figure 5f. Using the fabricated mold, one can get controlled and efficient synthesis of thousands of microchambers having a dimension of 150 × 150 × 60 μm3 (Figure 6a). Further reduction of size to 50 × 50 × 30 μm3 is possible (Figure 6c). Tailoring the dimensions depending on target cells and/or application is then achievable. High-throughput processing of up to 104 microchambers was imprinted on 10 × 10 mm2 area (Figure 6c). The versatility of the process allowed tailoring not only the X-Y dimensions but also the pillar height by controlling PG etching time, which then reflected the microchamber depths and aspect ratios. This is shown in Figure 5g,h, in which the brown color indicates that the modified regions were not completely removed. Considering an etching rate of 10 μm/min for our experimental data, the etching times of 3 min and 6 min produced 30-μm and 60-μm depth, respectively, which were much shallower than the thickness of modified regions, of more than 200 μm. By increasing the etching time, one can increase the structure depth. Multilayer modification Figurecan createFigure 5. Fabrication pillars5. Fabrication higher of PGof than PG glass glass 200 mold. μmold.m with PGPG aftercontrolled laser laser exposure lateral exposure dimensions.and and annealing annealing treatment: treatment: (a) sketch (a )of sketch of thethe irradiated irradiated patterns; patterns; (b) photograph photograph of ofthe the entire entire sample; sample; (c) optical (c) optical image of image central of region; central (d region;) (d) detaileddetailed view view of of (c ().c). PG PG afterafter chemicalchemical etching; etching; (e ()e 3D) 3D mold mold sketch; sketch; (f) photograph (f) photograph of the of entire theentire PG PG mold; (g) optical image of central region; (h) detailed view of (g). mold; (g) optical image of central region; (h) detailed view of (g).

Figure 6. SEM images of square microchambers obtained in PDMS by replication of the PG mold. (a) General view of the square microwells of 150 × 150 μm2 with 60-μm depth separated by a 50-μm thickness wall; (b) detailed view of (a); (c) general view of the square microwells of 50 × 50 μm2 with 30-μm depth separated by a 50-μm thickness wall; (d) detailed view of (c).

3.3. Fabrication of PG Masks with Controlled Dimensions for Additive Material Transfer Soft lithography is the most employed technique to create micro- and nanoscale patterns on solid substrates. Although providing the best resolution, the method is expensive, time-consuming, and involves sometimes up to tens of successive steps [39]. Alternative solutions for engineering smart biofunctional surfaces reported in literatures are microcontact printing [46], dip-pen nanolithography [40], ink-jet printing [47], and laser-induced forward transfer (LIFT) [48]. Each method presents distinct advantages, while also exhibiting specific limitations. Here we introduce the possibility to fabricate micropatterns on solid substrates by employing the mask imaging technique. We first employed PLAE to microfabricate hollow masks on PG using UV laser pulses. In this case, the main advantage of PLAE relies on the high versatility of laser direct Appl. Sci. 2020, 10, x FOR PEER REVIEW 10 of 18

Appl. Sci. 2020, 10, 8947 10 of 17

Using the fabricated mold, one can get controlled and efficient synthesis of thousands of microchambers having a dimension of 150 150 60 µm3 (Figure6a). Further reduction of size × × to 50 50 30 µm3 is possible (Figure6c). Tailoring the dimensions depending on target cells × × and/or application is then achievable. High-throughput processing of up to 104 microchambers was imprinted on 10 10 mm2 area (Figure6c). The versatility of the process allowed tailoring not only × the X-Y dimensions but also the pillar height by controlling PG etching time, which then reflected the microchamber depths and aspect ratios. This is shown in Figure5g,h, in which the brown color indicates that the modified regions were not completely removed. Considering an etching rate of 10 µm/min for our experimental data, the etching times of 3 min and 6 min produced 30-µ m and 60-µm depth, respectively,Figure 5. whichFabrication were of PG much glass mold. shallower PG after laser than exposure the thickness and annealing of treatment: modified (a) regions,sketch of of more than 200 µm. By increasingthe irradiated the patterns; etching (b) photograph time, one of canthe entire increase sample; the (c) structureoptical image depth. of central Multilayer region; (d) modification can create pillarsdetailed higher view of than (c). PG 200 afterµ mchemical with etching; controlled (e) 3D mold lateral sketch; dimensions. (f) photograph of the entire PG mold; (g) optical image of central region; (h) detailed view of (g).

Figure 6. FigureSEM 6. images SEM images of square of square microchambers microchambers obtained obtained in PDMS inPDMS by replication by replication of the PG mold. of the(a) PG mold. General view of the square microwells of 150 × 150 μm2 with 60-μm depth separated by a 50-μm (a) General view of the square microwells of 150 150 µm2 with 60-µm depth separated by a 50-µm thickness wall; (b) detailed view of (a); (c) general× view of the square microwells of 50 × 50 μm2 with thickness wall; (b) detailed view of (a); (c) general view of the square microwells of 50 50 µm2 with 30-μm depth separated by a 50-μm thickness wall; (d) detailed view of (c). × 30-µm depth separated by a 50-µm thickness wall; (d) detailed view of (c). 3.3. Fabrication of PG Masks with Controlled Dimensions for Additive Material Transfer 3.3. Fabrication of PG Masks with Controlled Dimensions for Additive Material Transfer Soft lithography is the most employed technique to create micro- and nanoscale patterns on solid Softsubstrates. lithography Although is the providing most the employed best resolution technique, the method to createis expensive, micro- time-consuming, and nanoscale and patterns involves sometimes up to tens of successive steps [39]. Alternative solutions for engineering smart on solidbiofunctional substrates. surfaces Although reported in literatures providing are microcontact the best printing resolution, [46], dip-pen the nanolithography method is expensive, time-consuming,[40], ink-jet and printing involves [47], and sometimes laser-induced up toforw tensard oftransfer successive (LIFT) [48] steps. Each [39 method]. Alternative presents solutions for engineeringdistinct advantages, smart biofunctional while also exhibiting surfaces specific reported limitations. inliteratures are microcontact printing [46], dip-pen nanolithographyHere we introduce [40 the], ink-jetpossibility printing to fabricate [47 ],micropatterns and laser-induced on solid substrates forward by transferemploying (LIFT) [48]. the mask imaging technique. We first employed PLAE to microfabricate hollow masks on PG using Each methodUV laser presents pulses. distinctIn this case, advantages, the main advantage while of also PLAE exhibiting relies on the specific high versatility limitations. of laser direct Here we introduce the possibility to fabricate micropatterns on solid substrates by employing the mask imaging technique. We first employed PLAE to microfabricate hollow masks on PG using UV laser pulses. In this case, the main advantage of PLAE relies on the high versatility of laser direct writing of almost any kinds of shapes. A representative example of round micropatterns with 200-µm diameter, formed in PG by LDW followed by annealing, is presented in Figure7a,b. The laser-irradiated micropatterns were constructed in volume using a lens with a 35-mm focal length and 40-mW power at an optimum scanning speed of 0.1-mm/s. This speed was precisely determined since, for slightly higher speeds, the programmed circle shape becomes elliptical. Two-layer exposure was employed by focusing the laser at 125 µm first and then 375 µm below the surface in a 500-µm-thick PG sample volume. This exposure can modify entire PG glass substrates along the z-axis so that the subsequent chemical etching treatment can create through-holes in the sample (Figure7c,d). Appl. Sci. 2020, 10, 8947 11 of 17

Appl. Sci. 2020, 10, x FOR PEER REVIEW 12 of 18

Figure 7. Fabrication of PG mask with microholes. PG mask after laser exposure and annealing Figure 7. Fabrication of PG mask with microholes. PG mask after laser exposure and annealing treatment: (a) photograph of the sample; (b) optical image of central region. PG mask after chemical treatment:etching: (a) photograph (c) photograph of of the the sample;sample; (d ()b optical) optical image image of central of centralregion, PG region. mask with PG microholes. mask after chemical etching: (c) photograph of the sample; (d) optical image of central region, PG mask with microholes.

In the second step, we employed additive MAPLE technique [3,49] to generate the micropatterns on silicon by transferring the evaporated material through the PG mask (Figure8). MAPLE is a physical deposition process, typically applied for assembling delicate compounds, both organic and inorganic, on solid substrates. Detailed theoretical and experimental aspects involved in MAPLE are presented elsewhere [50]. Brieﬂy, the procedure is based on laser evaporation of a frozen target, in which the material of interest is dissolved in a solvent. Typically, the ejected molecules are assembled on the substrate to deposit a continuous thin ﬁlm, while in this case, the structured PG glass was placed in Figure 7. Fabrication of PG mask with microholes. PG mask after laser exposure and annealing front of the substrate to generate micropatterns. As an additive process, the height of the patterns treatment: (a) photograph of the sample; (b) optical image of central region. PG mask after chemical could be easilyetching: controlled (c) photograph by of the the number sample; (d of) optical the laser image pulses of central applied region, forPG mask target with evaporation. microholes.

Figure 8. (a) Scheme of MAPLE (Matrix-Assisted Pulsed Laser Evaporation) process; (b) PG mask with microholes and silicon substrate after transfer of micropatterns; (c) SEM image of the transferred GON (Graphene Oxide Nanomaterials) micropatterns on silicon.

FigureFigure 8. (a )8. Scheme (a) Scheme of MAPLE of MAPLE (Matrix-Assisted (Matrix-Assisted Pulsed Pulsed Laser Laser Evaporation) Evaporation) process; ( (bb) )PG PG mask mask with microholeswith microholes and silicon and substrate silicon substrate after transfer after transfer of micropatterns; of micropatterns; (c) SEM(c) SEM image image of of the the transferredtransferred GON (GrapheneGON (Graphene Oxide Nanomaterials) Oxide Nanomaterials) micropatterns micropatterns on silicon. on silicon.

In Figure8b we show photographs of the PG mask after its use in MAPLE transfer of ejected molecules and silicon substrate on which desired patterns of materials are assembled. In this study, a mixture of organic precursors (phloroglucinol/glyoxylic acid/pluronic F127) was transferred on silicon through the PG mask, in order to generate different matrices of round micropatterns using optimized deposition conditions [51] that were shown to generate mesoporous carbon thin films. The separation distance between two adjacent micropatterns within each matrix was modified, and 100 structures were thus created in a single-step process. Graphene oxide nanomaterials (GON) were then transferred on silicon (Figure8c), in order to generate round micropatterns of 200 µm in diameter. We tested GON because recent advances in nanomedicine revealed extensive interests for fabricating graphene-based, cell-instructive microenvironments. Target applications are related to drug/gene delivery and cancer therapy, engineering stem cell responses, bacteria-killing and tissue-engineering platforms, biosensing, and cellular imaging [52]. Melanoma cells exposed to GON-BSA coatings with compositional gradient of inhibitors were found to exhibit a dose-dependent effect on target activity [13]. Model platforms of micropatterns having predesigned shapes and sizes could be further obtained by MAPLE using PG masks with different geometrical configurations in view of creating bioinstructive structures for various assays. PG masks could be viable alternatives to conventional metal stencil masks as the former may challenge the better resolution, sharper edges, and better uniformity of geometrical shape.

3.4. Mesenchymal Stem Cell Adhesion Studies on Laser-Processed Foturan Glass Surfaces Cell adhesion is a biological process of fundamental importance with implications in the correct development and cell-to-cell signaling and regulation. The mechanical interaction between a cell and its extracellular matrix (ECM) can influence and control cell behavior and function. In this study we tested the laser-processed PG surfaces from the point of view of cellular adhesion. We performed immunofluorescence, staining F-actin to evidence the cytoskeleton and the focal adhesion points with vinculin since they play key roles in the cell shape control. Specifically, actin filaments and focal adhesion contacts are commonly tested structures to assess cell adhesion on certain surfaces. The fluorescent staining was employed to demonstrate that no significant morphological changes were observed when comparing the surfaces of cover glass and processed glass. Generally, cytoskeletal structure and the cellular mechanical properties are determined by the cell–matrix and cell–cell adhesions. Many cellular functions including motility and cell division depend on the response to external mechanical forces, such as extracellular matrix and neighboring cells [53]. The capacity to generate and transmit forces depends on the strength of the assembly between the integrin adhesions’ receptors and the actin-myosin cytoskeleton mediated by focal adhesion proteins (as, e.g., vinculin). To test the biocompatibility of laser-processed PG surfaces, samples with and without collagen coating were used to analyze how cells interacted with them at two time intervals, 1 h and 24 h after seeding. The collagen coating was used, as it is known to support myogenic and osteogenic differentiation, MSCs being prone to commit to different phenotypes with extreme sensitivity to tissue-level elasticity [54]. For this purpose, we used MSCs grown on either cover glass controls or PG, bare or coated with collagen. As can be observed in Figure9, we noticed a slight di fference in the number of cells attached to the cover glass surface (Figure9a–d), with a slight increase of the number of cells grown on the noncoated surfaces (represented by “-”, Figure9a,c), irrespective of the time points. Although for this type of cell line, the number of attached cells is higher on bare cover glass, the collagen (represented by “+”, Figure9b,d) still provides a better adhesion, as it can be demonstrated by quantification of the degree of actin and vinculin colocalization (Figure9i). Same studies with MSCs grown on the laser-processed PG surfaces indicate a good biocompatibility supported by cell attachment tests (Figure9e,f) after 1 hour and by cell adhesion assays (Figure9g,h) after 24 h. We did not notice any difference in the number of attached cells (comparing 9e with 9f or 9g with 9h), while the increase in cell adhesion (Figure9j) was observed when collagen was used, similarly to studies carried out on the cover glass samples. We performed the colocalization analysis Appl. Sci. 2020, 10, 8947 13 of 17

toAppl. show Sci. that 2020, the 10, x laser-processed FOR PEER REVIEW glass did not induce changes in formation of focal adhesion14 of sites,18 which further played the role in mechanotransduction signaling (Figure9k).

FigureFigure 9. 9.Immunofluorescence Immunofluorescence microscopymicroscopy images of of MSCs MSCs (Mesenchymal (Mesenchymal Stem Stem Cells) Cells) grown grown on on covercover glass glass (a –(ad–)d and) and PG PG samples samples (similar (similar toto samplessamples presented in in Section Section 3.1.) 3.1.) developed developed by by PLAE PLAE (e–(he);–h bare); bare (a ,(ca,,ec,,ge),g or) or coated coated with with collagen collagen (b(b,d,d,f,f,h,h).). The The colocalization colocalization between between phalloidin phalloidin and and vinculinvinculin staining, staining, 1 1 h h post-seeding post-seeding waswas evaluatedevaluated by calculating colocalization colocalization coefficient coefficient using using JACoPJACoP (Just (Just Another Another ColocalizationColocalization Plugin) Plugin) and and the the corresponding corresponding results results are presented are presented in (i), ( inj), (andi), (j), and(k) ( k(mean) (mean of ofn n= =1414 ± SEM).SEM). Two-tailed Two-tailed Student’s Student’s t testt test was was used used to todetermine determine the the statistical statistical ± significance.significance. (** (**p pvalue value< <0.01, 0.01, ****** pp valuevalue < 0.001).0.001). Scale Scale bar bar isis 100 100 μm.µm. Appl. Sci. 2020, 10, 8947 14 of 17

With all devices described above, we can foresee some critical applications to provide cell culture-specific environments easily for investigation over long time intervals. Specifically, chemotaxis-induced cancer cell migration could be achieved over extended periods in diffusion-based gradient media. The 3D reconstruction of cell migration in a chip will give information on the locomotion mechanism in tight contact with narrow spaces and would provide the means to separate the most aggressive cancer cells for further analysis. The 3D characteristics are also essential for reproducing blood vessels, mimicking capillaries in a blood–brain barrier configuration for in vitro testing of microvascular permeability and validation of an Alzheimer disease model. In addition, further analysis using a microwell 3D geometry or 2D patterns allowing single cell trapping or controlling is biologically relevant as one may be able to assess, e.g., behavior of selected cells from the same population.

4. Conclusions PLAE technique was applied herein for the fabrication of different structures in PG and further proposed the fabricated structures for creation of biologically relevant microenvironments including: (1) large-scale area microfluidic channels for the development of a versatile perfusion system for cell culture chambers, (2) sharp-edge glass pillars for casting PDMS replica of thousands of microchambers made for single cell immobilization, and (3) PG masks with controlled geometric arrays of microholes for subsequent additive growth of biomolecules. In vitro tests evaluating mesenchymal stem cells’ behavior on the laser-processed PG surfaces demonstrated the cell viability, while a coating with collagen improved cellular adhesion. We consider PLAE an efficient alternative to FLAE for large-area laser processing and to conventional mask-based UV light exposure processes as it offers the versatility to fabricate any complex shapes either in volume or on PG surface. High repetition rate ps laser pulses allow, thus, a faster processing time and capability of developing complex surfaces and tridimensional environments with good biocompatibility at lower costs.

Author Contributions: Conceptualization, E.A., F.S., and K.S.; methodology, F.J., S.O., C.B., and M.Z.; investigation, F.J., S.O., M.Z., and E.A.; Writing, Original Draft Preparation, E.A. and F.S.; writing, review and editing, E.A., F.S., and K.S.; project administration, F.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by IFA (Institute of Atomic Physics) through ELI-RO_2020_11 project, no. 01/2020 and Romanian Ministry of Education and Research, under Romanian National Nuclei Program LAPLAS VI – contract no. 16N/2019. This work has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 871124 Laserlab-Europe. All authors have read and agreed to the published version of the manuscript. Acknowledgments: The authors are grateful to Gianina Popescu-Pelin for SEM technical support. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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